07 June 2011
UK scientists have developed a probe to monitor bicarbonate concentrations in mitochondria - components in living cells that generate chemical energy. Monitoring bicarbonate levels will improve researchers' understanding of its role in cellular reaction mechanisms.
A challenge when designing cellular probes is ensuring that the probe is not only selective for its target but can also be delivered to the site of interest within the cell. A team of scientists led by David Parker at the University of Durham has made a probe that can overcome this challenge.
The luminescent probe features an azaxanthone moiety, which is linked to a europium complex by an amide bond. The azaxanthone allows the probe's uptake into cells and localisation within the mitochondria, and the europium complex has an affinity for bicarbonate ions. The ability to probe bicarbonate levels 'can offer an unprecedented insight into signalling mechanisms', says Parker.
Bicarbonate ions are a product of the Krebs cycle - a series of enzyme-catalysed reactions that produce energy in living cells. The ions arise from carbon dioxide generated by the Krebs cycle. The team tested the probe on HeLa cells in an incubation chamber. When they raised the level of carbon dioxide in the chamber, the bicarbonate ion concentration in the mitochondria increased, causing the luminescence signal to grow stronger. The effect was reversed when they decreased the carbon dioxide level.
Images of HeLa cells, showing the stained mitochondrial regions at carbon dioxide levels of 3, 4 and 5 per cent (left to right)
The team developed the probe further to provide a ratiometric response to analyse changes in signal intensities in cells. They found that the intensity did not change with bicarbonate levels when the europium ion was substituted with terbium, so they suggest that this provides an internal calibration system when the two probes are used in tandem.
'The key point is the fact that by using macropinocytosis [cellular ingestion process in which the plasma membrane folds inwards to bring substances into the cell], several subcellular localisations could be targeted,' says Anne-Sophie Chauvin, an expert in the use of lanthanide complexes for biological applications at the Federal Polytechnic Schoolof Lausannein Switzerland. 'These findings open good perspectives for cellular monitoring and ratiometric analysis.'
Parker's team is working towards being able to monitor changes in bicarbonate concentrations in real-time and they plan to study the role of bicarbonate ions in vivo.